Synaptic vesicle pools at the frog neuromuscular junction

We have characterized the morphological and functional properties of the readily releasable pool (RRP) and the reserve pool of synaptic vesicles in frog motor nerve terminals using fluorescence microscopy, electron microscopy, and electrophysiology. At rest, about 20% of vesicles reside in the RRP, which is depleted in about 10 s by high-frequency nerve stimulation (30 Hz); the RRP refills in about 1 min, and surprisingly, refilling occurs almost entirely by recycling, not mobilization from the reserve pool. The reserve pool is depleted during 30 Hz stimulation with a time constant of about 40 s, and it refills slowly (half-time about 8 min) as nascent vesicles bud from randomly distributed cisternae and surface membrane infoldings and enter vesicle clusters spaced at regular intervals along the terminal. Transmitter output during low-frequency stimulation (2-5 Hz) is maintained entirely by RRP recycling; few if any vesicles are mobilized from the reserve pool.     

Synaptic restructuring during long-term facilitation at the crayfish neuromuscular junction

Long-term facilitation was induced by 20-Hz stimulation of the motor axon innervating the opener muscle of the crayfish, Procambarus clarkii. Excitatory postsynaptic potentials remained potentiated for several hours after stimulation. Structural correlates of potentiation were sought. Nerve terminals of the motor axon were fixed for electron microscopy in unstimulated preparations (controls), and during and after 20-Hz stimulation. Synapses were reconstructed from micrographs obtained from serial sections. Synaptic contact area and the number of vesicles at the presynaptic membrane did not change after 20-Hz stimulation, but the latter decreased during stimulation. Presynaptic dense bars ("active zones") decreased in number during and increased after stimulation, while perforated synapses increased after stimulation. Modification of presynaptic structures occurs rapidly and may be linked to long-lasting changes in quantal content of transmission.     

Two synaptic vesicle pools,vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions (DNMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K⁺. The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca²⁺. Mobilization of internal Ca²⁺ stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and DNMJs are suitable for further studies.     

Synaptic plasticity at the crayfish opener neuromuscular preparation

The crayfish opener neuromuscular preparation exhibits most of the plasticities yet described for any synapse, including facilitation, long-term potentiation, presynaptic inhibition, and modulation. Since the presynaptic terminals and postsynaptic muscle fibers can both be intracellularly penetrated, one can now more easily examine the cellular/molecular bases for these plasticities. Data from such studies suggest that facilitation may be influenced by something other than residual free calcium and that presynaptic inhibition is produced by a conductance increase to chloride in the terminals of the excitor axon. Several drugs (ethanol, pentobarbital) have significant effects on these synaptic plasticities over concentration ranges which produce obvious behavioral effects in crayfish and mammals. Hence, this preparation should be a useful model system to determine cellular/molecular bases for various synaptic plasticities and the effects of drugs on these plasticities.     

GABA inactivation at the crayfish neuromuscular junction

Changes in the effective membrane resistance of the abductor muscle of the dactylopodite of the crayfish were used to indicate changes in the GABA concentration in the synaptic cleft. Following bath application of GABA (10^?5 to 5 × 10^?5M), the muscle membrane resistance decreased and then increased slowly over the next few minutes. Renewing the solution or stirring the bath restored the GABA effect. Higher GABA concentrations produced a large stable decrease in membrane resistance. An active uptake system for GABA in the junctional region is suggested by the observation that the slow increase in membrane resistance following GABA application was decreased by cooling to 2°C or by the addition of known GABA uptake blockers such as L -DABA, β-guanidinopropionic acid, or nipecotic acid. The transport inhibitors, PCMBS and chlorpromazine, produced irreversible decreases in muscle membrane resistance, which precluded examining their effects on GABA inactivation. The decrease in GABA effect was not dependent on the external sodium concentration or on the degree of receptor activation. Nipecotic acid, which blocked GABA inactivation, did not affect the decay of the neurally evoked inhibitory junctional potential.     

Maximum likelihood estimation of non-uniform transmitter release probabilities at the crayfish neuromuscular junction

The classical model of quantal release of neurotransmitter assumes that a fixed number of quantal units are available for release in the presynaptic terminal, and that each unit has the same probability of being released. This model also assumes that different units are released independently of one another. We consider two variations of the classical model. In the first case we assume that release is independent, but with potentially different release probabilities at different sites. In the second case we allow for dependence among the release units. A maximum likelihood procedure for the estimation of model parameters is developed, and an estimator of the number of quantal units is proposed. The performance of the method is assessed through a simulation study, and the procedures are applied to the analysis of a sequence of post-synaptic potentials recorded intracellularly at the crayfish neuromuscular junction. Goodness of fit and hypothesis test procedures reject the classical model in favor of an independent release mechanism with differing release probabilities. A more general release mechanism, allowing for dependence in the release process, also provides a good fit to the data analyzed.     

In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction

Recently, it has become possible to directly detect changes in neuropeptide vesicle dynamics in nerve terminals in vivo and to measure the release of neuropeptides induced experimentally or evoked by normal behavior. These results were obtained with the use of transgenic fruit flies that express a neuropeptide tagged with green fluorescent protein. Here, we describe how vesicle movement and neuropeptide release can be studied in the larval Drosophila neuromuscular junction using fluorescence microscopy. Analysis methods are described for quantifying movement based on time lapse and fluorescence recovery after photobleaching data. Specific approaches that can be applied to nerve terminals include single particle tracking, correlation and Fourier analysis. Utilization of these methods led to the first detection of vesicle mobilization in nerve terminals and the discoveries of activity-dependent capture of transiting vesicles and post-tetanic potentiation of neuropeptide release. Overall, this protocol can be carried out in an hour with ready Drosophila.     

Nitric oxide and cyclic GMP induce vesicle release at Drosophila neuromuscular junction

Nitric oxide (NO) diffuses as short‐lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO‐induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO‐sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO‐induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca²⁺‐free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca²⁺‐independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 337–346, 1999     

Localization effects of acetylcholine release from a synaptic vesicle at the neuromuscular junction.

A two-dimensional compartment model devised for the appropriate representation of the transient process of the spontaneous generation of miniature endplate current (MEPC) at the neuromuscular junction is applied for clarifying the biochemical significance of the quantal release mechanism of acetylcholine (ACh), a typical neurotransmitter, in the synaptic chemical transmission process. The simulation analysis with the model demonstrates that the localization of the ACh release due to the fusion of a synaptic vesicle with the presynaptic membrane has significant effects on the amplitude of MEPC and that the stronger effects are caused with the smaller diffusion coefficients of ACh in the cleft. The sharpest and highest response of MEPC is achieved when the release area is about 4 times to the natural release through the narrow pore. On the other hand, the actual localization corresponding to the natural release of ACh makes the amplitude of MEPC higher by a factor about 2.5 compared with that in the most extended release of ACh examined, implying that the natural release mechanism works as an amplifier of the MEPC with the fixed amount of ACh available.     

Nitric oxide and cyclic GMP induce vesicle release at Drosophila neuromuscular junction.

Nitric oxide (NO) diffuses as short-lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO-induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO-sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO-induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca2+-free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca2+-independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae.     

Maintained depolarization of synaptic terminals facilitates nerve-evoked transmitter release at a crayfish neuromuscular junction

Presynaptic and postsynaptic potentials were examined by intracellular recording at a crayfish neuromuscular junction. During normal synaptic transmission, the action potentials were recorded in the terminal region of the excitatory axon and postsynaptic responses were obtained in the muscle fibers. We found that it was possible to modify the synaptic transmission by applying depolarizing or hyperpolarizing currents through the presynaptic intracellular electrode. Typically, a 7-15 mV depolarization lasting longer than 50 msec leads to a large (500%) enhancement of transmitter release, even though the preterminal action potential is reduced in amplitude. Hyperpolarization increases the amplitude of the action potential, but slightly reduces the transmitter release. These results are different from those reported for other neuromuscular synapses and the squid giant synapse, but are similar in many respects to the results reported for several invertebrate central synapses. We conclude, first, that different synapses may have markedly different responses to conditioning by membrane polarization and, secondly, that maintained low-level depolarization may induce a potentiated state in the nerve terminal, perhaps brought about by slow entry of calcium.     

Synaptic dynamics at the neuromuscular junction: mechanisms and models

During development, the neuromuscular junction passes through a stage of extensive polyinnervation followed by a period of wholesale synapse elimination. In this report we discuss mechanisms and interactions that could mediate many of the key aspects of these important developmental events. Our emphasis is on (1) establishing an overall conceptual framework within which the role of many distinct cellular interactions and molecular factors can be evaluated, and (2) generating computer simulations that systematically test the adequacy of different models in accounting for a wide range of biological data. Our analysis indicates that several relatively simple mechanisms are each capable of explaining a variety of experimental observations. On the other hand, no one mechanism can account for the full spectrum of experimental results. Thus, it is important to consider models that are based on interactions among multiple mechanisms. A potentially powerful combination is one based on (1) a scaffold within the basal lamina or in the postsynaptic membrane which is induced by nerve terminals and which serves to stabilize terminals by a positive feedback mechanism; (2) a sprouting factor whose release by muscle fibers is down-regulated by activity and perhaps other factors; and (3) an intrinsic tendency of motor neurons to withdraw some connections while allowing others to grow.     

Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.     

Cyclic AMP, 5-HT, and the modulation of transmitter release at the crayfish neuromuscular junction

In this study it was found that several agents which elevate cAMP levels in cells also increase dramatically the quantity of transmitter released from crayfish excitatory nerve terminals in response to a stimulus. With respect to time course and magnitude, the increase produced by one of these agents, the cyclic nucleotide phosphodiesterase inhibitor Squibb 20,009 (SQ 20,009), is unlike any reported for such a drug at a synapse. Additionally, SQ 20,009 potentiated the facilitation of transmitter release produced by serotonin (5-HT) at this synapse. These results establish a possible role for cAMP in the control and modulation of transmitter release at the crayfish neuromuscular junction (NMJ). They further suggest that 5-HT functions here by activation of a presynaptically located adenylate cyclase.     

Synaptic facilitation in Aplysia explored by random presynaptic stimulation

The identified interneuron L10 in the abdominal ganglion of Aplysia was stimulated to fire action potentials in a random sequence while the early inhibitory potential of its follower cell L2 was recorded. Application of Wiener nonlinear analysis to these data yielded a predictive model of the facilitating postsynaptic potential. The model shows that facilitation changes both the time-course and the magnitude of the early synaptic potential. The facilitated response has a longer duration than the unfacilitated response. Its magnitude is exponentially decreasing with increasing interstimulus interval between test and conditioning stimuli. Facilitation is abolished at short interstimulus intervals. The hypothesis that the magnitude only of transmitter release is increased cannot explain these results. The observed facilitation may be due to characteristics of pre- and postsynaptic morphology.     

Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction

Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.     

Glutamate inhibitors in the crayfish neuromuscular junction

1. The effects of chlorisondamine and TI-233 on the crayfish neuromuscular junction were investigated in order to compare the action of glutamate with that of the excitatory transmitter. 2. The glutamate-induced synaptic current was inhibited by both of these two drugs. Excitatory junctional potentials were significantly reduced by chlorisondamine, whereas they were increased by TI-233. 3. It is suggested that chlorisondamine and TI-233 are powerful non-competitive antagonists for glutamate. 4. A quantum analysis of extracellular EJPs demonstrated that chlorisondamine did not possess presynaptic action in the crayfish neuromuscular junction. Chlorisondamine shortened the decay phase of extracellular EJPs, and the decay was frequently fitted by a double exponential in relatively low concentrations. 5. Semilogarithmic plots of the decay phase of the glutamate current evoked by a short glutamate pulse were nearly linear, but they shifted from linearity to some extent in the presence of chlorisondamine, showing prolongation of the glutamate current tails. 6. When TI-233 was added to the bathing solution at a concentration of 0.1 mM, the quantum content of extracellular EJPs was increased by about two times, but the average unit size was not changed. 7. There was no change in the rise time and the decay phase of the glutamate potential in the presence of TI-233. 8. Pharmacological difference between glutamate responses and EJPs was revealed in the presence of chlorisondamine and TI-233. Unless this difference can be explicated with a reasonable explanation on the glutamate transmitter hypothesis, it is difficult to confirm that glutamic acid is an excitatory transmitter at the crayfish neuromuscular junction.